A large number of peptides have been approved for use in medical practice, and the peptides may be produced in suitable host cells by recombinant DNA technology or they may be produced synthetically by well established peptide synthesis technology. However, native peptides as well as analogues thereof tend to exhibit high clearance rates which are unacceptable for many clinical indication where a high plasma concentration of the peptide is required over a prolonged period of time. Examples of peptides which in their native form have a high clearance are: ACTH, corticotropin-releasing factor, angiotensin, calcitonin, insulin, glucagon, glucagon-like peptide-1 (GLP-1), glucagon-like peptide-2 (GLP-2), insulin-like growth factor-1, insulin-like growth factor-2, gastric inhibitory peptide, growth hormone-releasing factor, pituitary adenylate cyclase activating peptide, secretin, enterogastrin, somatostatin, somatotropin, somatomedin, parathyroid hormone, thrombopoietin, erythropoietin, hypothalamic releasing factors, prolactin, thyroid stimulating hormones, endorphins, enkephalins, vasopressin, oxytocin, opioids and analogues thereof, superoxide dismutase, interferon, asparaginase, arginase, arginine deaminase, adenosine deaminase and ribonuclease.
A variety of derivatizations of peptides and peptide analogs have been found to influence the clearance rate of the peptides in a favourable direction. One such derivatization is the introduction of a lipophilic acyl group into the therapeutic peptide causing a desirable protracted profile of action relative to the non-acylated peptide. Hence, less frequent administration of the therapeutic protein improves the patients compliance to the prescribed therapy, and it reduces the amount of peptide to be administered. This has been described and demonstrated in WO98/08871, which i.a. discloses acylation of GLP-1 and analogs thereof, in WO98/08872, which i.a. discloses acylation of GLP-2 and analogs thereof, and WO99/43708, which i.a. discloses acylation of exendin and analogs thereof. Mono- or dipeptide spacers such as aspartic acid and glutamic acid, between the peptide and the acyl-group was demonstrated to be desirable. Spacers including a free carboxylic acid group must be protected before acylation and subsequently deprotected.
EP 1227107 discloses the acylation of ∈-amino groups of human insulin.
WO00/55119 discloses a method for acylating peptides (e.g. GLP-1) and novel acylating agents.
In order for therapeutic peptides to be economically viable the cost of producing the peptides as well as the therapeutic dosage of the peptide are pivotal. A major cost during production of therapeutic peptides is the purification steps required to separate the target protein from impurities which are closely related to the target protein, e.g. isomers, desamido forms etc. These purification steps are usually performed by chromatography implying expensive chromatography matrices and solvents as well as reduced overall yield.
It is the aim of the present invention to provide an efficient and economic method for the introduction of lipophilic groups into peptides via α-amino-α,ω-dicarboxylic acid spacers. The method is more specific, and thus results in higher yields and reduced formation of closely related impurities. A significant reduction of the cost of producing the acylated peptides are achieved. Less expensive acylated peptides are highly desirable for maximizing the number of patients for whom the treatment is available as well as for exploiting the advantages of alternative delivery routes which have lower bioavailability than subcutaneous injection, e.g. transdermal and pulmonal delivery.